Abstract Changes in fructose uptake have been implicated in cancers development, and transition into more aggressive phenotypes. Many efforts have been directed to use GLUTs as therapeutic targets to distinguish cancer cells for disease diagnosis and treatment. However, specific targeting of disease-relevant GLUTs is an unmet need. We hypothesize that targeting the composition and activity of disease-relevant GLUTs will enable rapid and accurate identification of metabolic alterations and disease diagnosis and therapy. However, a significant limitation hampers the GLUT-based cell identification. That is the absence of molecular tools to discriminate between GLUTs. Thus, it is our goal to enable specific targeting of different GLUTs for effective discrimination between normal and cancer cells and between cancer subtypes through the following specific aims: Aim 1. Exploring the cargo capacity of fructose GLUTs with fluorophores. We propose to carry out a systematic exploration of cargo preferences for two major fructose transporters ? GLUTs 2 and 5 - using fluorophores as cargoes and simultaneous real-time reporters of the uptake. Understanding the differences that exist between GLUTs will directly promote the development of GLUT-specific analytical and biomedical tools. Aim 2. Exploring the impact of sugar conformation on substrate selection by fructose transporters. Fructose transport in cells is facilitated by GLUT5 and non-specific GLUTs 2, 7, 9, 11 and 12. While significant literature addresses substrate selection by GLUT5, the substrate-uptake relationship for other transporters and their relevance to cancer is not well understood. Within this aim, the ability of GLUT2 and GLUT5 to discriminate between sugar conformations will be assessed through focused structure-uptake relationship studies. The acquired knowledge will lay the foundation for designing molecular probes to target fructose-specific vs. fructose- nonspecific GLUTs. Aim 3. Employing small molecule-based GLUT analysis in cancer cell discrimination. The significant difference in fructose uptake between normal and cancer cells supports quantitative analysis of fructose uptake as a measure of carcinogenesis. The focus of this aim is to explore whether targeted analysis of GLUT5 and cell metabolism can provide effective approach to identify metabolically-compromised cells such as cancer. The approach is based on using GLUT-specific molecular probes. The knowledge, probes, and analytical approaches produced herein will lay foundation for the development of diagnostic agents and, possibly, medicinal agents as regulators of sugar uptake and cellular metabolism. The outlined research plan is also geared to promote the education of next generations of scientists as the program heavily involves researchers at undergraduate, graduate, and postdoctoral levels. The proposed research project directly contributes to fulfilling the mission of NIH AREA award and strengthen the research and an educational environment in health-related fields at Michigan Tech.